Our long-term aim is to elucidate the cellular and molecular events underlying the early ontogeny of the hematopoietic system in the mammalian embryo. While definitive hematopoiesis in the fetus and adult is known to be multilineage, primitive hematopoiesis in the yolk sac is centered on the synthesis of erythroid cells that are critical for survival of the embryo. We recently made the seminal observation that primitive erythroblasts in the mouse embryo ultimately enucleate. However, the process and mechanisms regulating enucleation of primitive erythroid cells remain unknown and are the subject of this renewal. In the first aim of this proposal, we will test the hypothesis that primitive erythroblasts respond to chemotactic stimuli, including SDF-1, that facilitate the migration of hematopoietic stem and progenitor cells to the fetal liver. In the second aim, we will test the hypothesis that primitive erythroid cells can and do interact with fetal liver macrophage cells, using erythroblast island reconstitution and enucleation assays established in the laboratory. The adhesion molecules emp and ICAM4 each play important roles in definitive erythroid-macrophage interactions. We have obtained preliminary data that primitive erythroblasts express both emp and ICAM4 and in the third aim we will test the hypothesis that these molecules are functionally important for terminal maturation of primitive erythroblasts. Finally, we have recently discovered that primitive erythroid cells in mice lacking c-myb fail to enucleate and in the fourth aim we will test the hypothesis that this enucleation failure is erythroid cell non-autonomous. These studies of terminal erythroid maturation are facilitated by the synchronous differentiation and enucleation of primary primitive erythroid cells. Furthermore, these studies will allow us to compare and contrast the terminal maturation of primitive erythroid cells with definitive erythroid cells that mature extravascularly in erythroblast islands. A better understanding of the mechanisms responsible for terminal erythroid maturation will ultimately lead to the efficient and complete ex vivo synthesis of erythrocytes to serve an ever-expanding clinical need for red blood cells.